Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum.

CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3' UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR- mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.

Microbiomes of Various Maternal Body Systems Are Predictive of Calf Digestive Bacterial Ecology.

Body systems once thought sterile at birth instead have complex and sometimes abundant microbial ecosystems. However, relationships between dam and calf microbial ecosystems are still unclear. The objectives of this study were to (1) characterize the various maternal and calf microbiomes during peri-partum and post-partum periods and (2) examine the influence of the maternal microbiome on calf fecal microbiome composition during the pre-weaning phase. Multiparous Holstein cows were placed in individual, freshly bedded box stalls 14 d before expected calving. Caudal vaginal fluid samples were collected approximately 24 h before calving and dam fecal, oral, colostrum, and placenta samples were collected immediately after calving. Calf fecal samples were collected at birth (meconium) and 24 h, 7 d, 42 d, and 60 d of age. Amplicons covering V4 16S rDNA regions were generated using DNA extracted from all samples and were sequenced using 300 bp paired end Illumina MiSeq sequencing. Spearman rank correlations were performed between genera in maternal and calf fecal microbiomes. Negative binomial regression models were created for genera in calf fecal samples at each time point using genera in maternal microbiomes. We determined that Bacteroidetes dominated the calf fecal microbiome at all time points (relative abundance ≥42.55%) except for 24 h post-calving, whereas Proteobacteria were the dominant phylum (relative abundance = 85.10%). Maternal fecal, oral, placental, vaginal, and colostrum microbiomes were significant predictors of calf fecal microbiome throughout pre-weaning. Results indicate that calf fecal microbiome inoculation and development may be derived from various maternal sources. Maternal microbiomes could be used to predict calf microbiome development, but further research on the environmental and genetic influences is needed.

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism.

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

Identification of RECK as an evolutionarily conserved tumor suppressor gene for zebrafish malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are a type of sarcoma with poor prognosis due to their complex genetic changes, invasive growth, and insensitivity to chemo- and radiotherapies. One of the most frequently lost chromosome arms in human MPNSTs is chromosome 9p. However, the cancer driver genes located on it remain largely unknown, except the tumor suppressor gene, p16 (INK4)/CDKN2A. Previously, we identified RECK as a tumor suppressor gene candidate on chromosome 9p using zebrafish-human comparative oncogenomics. In this study, we investigated the tumorigenesis of the reck gene using zebrafish genetic models in both tp53 and ribosomal protein gene mutation background. We also examined the biological effects of RECK gene restoration in human MPNST cell lines. These results provide the first genetic evidence that reck is a bona fide tumor suppressor gene for MPNSTs in zebrafish. In addition, restoration of the RECK gene in human MPNST cells leads to growth inhibition suggesting that the reactivation of RECK could serve as a potential therapeutic strategy for MPNSTs.